Riboflavin overproduction on lignocellulose hydrolysate by the engineered yeast Candida famata

Abstract Lignocellulose (dry plant biomass) is an abundant cheap inedible residue of agriculture and wood industry with great potential as a feedstock for biotechnological processes. Lignocellulosic substrates can serve as valuable resources in fermentation processes, allowing the production of a wide array of chemicals, fuels, and food additives. The main obstacle for cost-effective conversion of lignocellulosic hydrolysates to target products is poor metabolism of the major pentoses, xylose and L-arabinose, which are the second and third most abundant sugars of lignocellulose after glucose. We study the oversynthesis of riboflavin in the flavinogenic yeast Candida famata and found that all major lignocellulosic sugars, including xylose and L-arabinose, support robust growth and riboflavin synthesis in the available strains of C. famata. To further increase riboflavin production from xylose and lignocellulose hydrolysate, genes XYL1 and XYL2 coding for xylose reductase and xylitol dehydrogenase were overexpressed. The resulting strains exhibited increased riboflavin production in both shake flasks and bioreactors using diluted hydrolysate, reaching 1.5 g L−1.


Introduction
Riboflavin (vitamin B 2 ) is a water-soluble vitamin, produced by all plants and most of micr oor ganisms.It is essential for growth and r epr oduction of humans and animals .Ribofla vin is the precursor of flavin nucleotides (FMN and FAD), crucial coenzymes involved in various o xidoreducti ve reactions (Abbas andSibirny 2011 , Sc hwec hheimer et al. 2016 ).Riboflavin is an important biotec hnological pr oduct that is used mainly in a gricultur e as a feed ad diti ve, as well as in the food industry and medicine (Beztsinna et al. 2016, You et al. 2021 ).Riboflavin is curr entl y biotec hnologicall y pr oduced by engineer ed str ains of the bacterium Bacillus subtilis and the filamentous fungus , Ashb y a (Eremothecium) gossypii (Ruchala et al. 2022 ) .To enhance microbial riboflavin pr oduction, str ains wer e impr ov ed thr ough metabolic engineering and classical selection (Zhao et al. 2021 ).These appr oac hes involv ed r andom m uta genesis induced by c hemical exposure and UV irradiation, as well as random and site-directed m uta genesis ac hie v ed thr ough geneticall y engineer ed deletions, insertions, or substitutions (Zhao et al. 2021 ).Additionally, the fermentation process was optimized by selecting and adjusting medium components and their concentrations (You et al. 2021 ).
Ho w e v er, further r esearc h is r equir ed to enhance industrial ribofla vin processes , focusing on impro ving k e y ste ps, including fermentation conditions, purification techniques, and the utilization of recycled sources .T he price of riboflavin depends significantly on the price of the carbon substrate used (Kato and Park 2012 ).It would be especially important to use waste as a carbon source.
In our current w ork, w e studied riboflavin production by the yeast Candida famata (teleomorph form is known as Debaryomyces subglobosus Nguyen et al. 2009 ) .T he ribofla vin o v er pr oducing strain of C. famata was used for more than 10 years for industrial riboflavin production.Ho w ever, its production w as discontinued due to genetic instability of the used strain (Abbas and Sibirny 2011 ).We constructed stable non-r e v erting riboflavinov er pr oducing str ains of C. f amata using a combination of classical selection methods and metabolic engineering.These strains accumulated 1-1.45 g L −1 of riboflavin in flasks and r eac hed 16.4 g L −1 in a 7 L bioreactor during fed-batc h cultiv ation (Dmytruk et al. 2011, Dmytruk et al. 2014 ).A yeast str ain ov er expr essing the RFE1 gene, which encodes riboflavin excr etase, pr oduced 1.7 g L −1 of vitamin B 2 (Tsyrulnyk et al. 2020 ).Modulation of the purine biosynthesis pathway further enhanced riboflavin pr oduction, r esulting in up to 2.85 g L −1 (Dmytruk et al. 2020 ).The engineered str ains pr oduced riboflavin in media with glucose as the carbon source .T he latest str ain, expr essing Sef1-a positiv e r egulator of riboflavin synthesis-under a lactose-inducible promoter, or the structural RIB6 gene, achieved riboflavin production of approximately 2.5 g L −1 using milk whey, a byproduct of cheese production, as the carbon source (Ruchala et al. 2022 ).
Lignocellulosic hydr ol ysates can serv e as v aluable r esources for fermentation processes, allowing the production of a wide r ange of c hemicals , fuels , and food ad diti ves .For instance , ethanol (Robak and Balcerek 2020 ), organic acids (Jimenez-Quero et al. 2020), pol ymers (Kawa guc hi et al. 2017), and enzymes (Namnuch et al. 2021 ) are among the diverse products that can be synthesized through this approach.Different yeast species have been considered for the production of chemicals, mainly ethanol and xylitol, using different lignocellulosic substrates, such as horticultural waste olive tree pruning, rice straw, corncob, and sugarcane ba gasse (Ber gmann et al. 2019 ).
In this study, we demonstrated that engineered strains of C. famata exhibit riboflavin ov er pr oduction in medium containing lignocellulose hydr ol ysate .We in v estigated both, pr e viousl y iso-lated, and ne wl y constructed r ecombinant str ains with ov er expression of XYL1 and XYL2 genes encoding for xylose reductase and xylitol dehydr ogenase, r espectiv el y, for their riboflavin production capabilities in flask and bioreactor experiments.

Strains, media, and cultivation conditions
C. famata VKMY-9 (wild type) and BRP/PRS3m/ADE4 m (designated as BRPI from the Best Riboflavin Producer Improved) (Dmytruk et al. 2020 ) strains were used in this study.Riboflavin oversynthesis by BRPI is independent of iron ions.Yeast strains were cultivated at 30 • C on rich YPD (5 g L −1 yeast extract, 10 g L −1 peptone, and 20 g L −1 glucose) or mineral YNB (1.7 g L −1 yeast nitrogen base, 5 g L −1 ammonium sulphate and 20 g L −1 glucose or other carbon sources) media.The modified YNB medium (containing 1.7 g L −1 yeast nitrogen base, 3 g L −1 ammonium sulfate, and 2 g L −1 Ta ble 1. Gro wth and riboflavin production of the C. famata BRPI strain in YNB medium supplemented with different sources of carbon: Cultivation time-120 hours.
DH5 α w as gro wn at 37 • C in LB medium as described (Sambrook et al. 1989 ). Transformed E. coli cells were maintained in rich medium containing 100 mg L −1 of ampicillin.
To estimate riboflavin synthesis the yeast cells from a fresh plate wer e gr own in 50 mL of liquid media in 250 mL Erlenmeyer flasks with initial biomass 5 mg L −1 .To pr epar e for bior eactor cultiv ation, a pr ecultur e of C. f amata str ains was incubated with shaking at 220 rpm and 28 • C for 24 h in YPD.This pr ecultur e was used to inoculate the bioreactors with a starting volume of 500 mL of batch medium and a starting biomass of 1 g CDW L −1 .Fermentations were carried out in 1 Liter total volume bioreactors (Sartorius Biostat ® B2).The fermentation temper atur e was maintained at 28 • C, the pH was controlled at 5.5 ± 0.5 by adding 100 g L −1 sodium hydroxide, and the dissolved oxygen concentration was

Plasmids and strains constructions
Standard cloning techniques were used as described (Sambrook et al. 1989 ).Genomic DNA of C. famata was isolated using the NucleoSpin ® Tissue Kit (Mac her ey-Na gel, Dur en, German y).Restriction endonucleases and DNA ligase (Thermo Fisher Scientific Baltics , Vilnius , Lithuania) were used following the manufacturer's specifications.Plasmid isolation from E. coli was performed with the ZyppyTM Plasmid Miniprep (Irvine , C A, USA).PCR amplification of the fr a gments of inter est was performed using Phusion High-Fidelity DNA Pol ymer ase (T hermo Fisher Scientific Baltics , Vilnius, Lithuania) according to the manufacturer's specification.PCRs were performed in a GeneAmp PCR System 9700 thermocycler (Applied Biosystems, Foster City, CA, USA).
The nourzeotricin resistance gene SAT-1 was amplified from the pTb/SAT-1/R1 (Petrovska et al. 2022 )  The XYL1 gene was amplified from genomic DNA of VKMY-9 using the pair Ko1260 (CGC GGA TCC A TG TCT A TT AAG TTG AAT TCA GGA TAT G)/Ko1261 (AAA CTG CAG TTA AGC AAA GAT TGG AAT CTT GTC C).The BamHI and PstI restriction sites were incor por ated into the Ko1260 and Ko1261 r espectiv el y.After the BamHI/PstI restriction the XYL1 was cloned between TEF1 promoter and terminator into the corresponding sites of pT-SAT to create pT -SAT -X1 (Fig. 1 A).
The XYL2 gene was amplified from genomic DNA of VKMY-9 using a pair of primers Ko1359 (CGC GGA TCC ATG ACA CCA AAC CCT TCC TTA G)/Ko1360 (AAA CTG CAG TTA TTC TGG A CC A CT GA T GA T AC).The BamHI and PstI restriction sites were incorporated into primers Ko1359 and Ko1360 r espectiv el y.After the BamHI/PstI restriction the XYL2 was cloned between TEF1 promoter and terminator into the corresponding sites of pT-SAT to create pT -SAT -X2.The XYL2 gene with TEF1 promoter and terminator was amplified using the pair Ko1362 (CCG CTC GAG AAA TTG ACT GGT CTG AAA TAA TAG)/Ko1363 (CCG CTC GAG ATG TTG CGC CGA ACA ATC AC), treated with XhoI and cloned into XhoI-linearized pT -SAT -X1 to create pT -SAT -X1/X2 (Fig. 1 C).
Plasmids pT -SAT -X1 and pT -SAT -X1/X2 were linearized with the restriction endonuclease AatII and used for transformation of the BRPI.The transformants were selected on a solid mineral medium, containing nourseothricin at 20 mg L −1 on the third day of incubation.Subsequently, the selected strains Table 2. Cell biomass , ribofla vin production and riboflavin yield of the r ecombinant str ains BRPI/XYL1 and BRPI/XYL1/XYL2 of C. famata on YNB medium with xylose r epr esented at 96 h of culti vation.Relati ve expression levels of the XYL1 and XYL2 genes in the BRPI/XYL1 and BRPI/XYL1/XYL2 str ains v ersus the BRPI recipient strain.Relative expression levels were obtained using the compar ativ e Ct method for quantification of the BRPI/XYL1 and BRPI/XYL1/XYL2 were checked by PCR using a pair of primers Ko977 (CCC AAG CTT AAA TTG ACT GGT CTG AAA T AA T AG)/Ko1261 to verify the presence of the expression module of XYL1 (Fig. 1 B, D) and K o1356/K o1361 to verify of presence the expression module of XYL2 (Fig. 1 E).The selected transformants were stabilized through cultivation in non-selective media for 12-14 generations, follo w ed b y a shift to selective media using replica plating.The same round of stabilization was repeated after the stor a ge of the selected strains at -80 • C for three months .T he presence of corresponding expression modules in the stabilized transformants was confirmed via PCR using the same primers.

Quantitati v e Real-Time PCR
The expression of the XYL1 gene was confirmed by qRT-PCR.Total RN A w as extr acted fr om yeast cells using the GeneMATRIX Universal RNA Purification Kit with DNAseI (EURx Ltd., Gdansk, P oland).T he qR T-PCR w as performed b y 7500 Fast Real-Time PCR System (The Applied Biosystems, USA) with a SG OneStep qRT-PCR kit (EURx Ltd., Gdansk, Poland) using corresponding pairs of primers XYL1_Cf_f_qRT (ATG TCT ATT AAG TTG AAT TCA GGA T A T)/XYL1_Cf_r_qRT (TT A AGC AAA GA T TGG AA T CTT GTC), XYL2_f_Cf (GTG GAC GCC A T A TTC AAA TTG)/XYL2_r_Cf (GTC A T A AGC GTC AAT AGC TTC), and ACT1f (TAA GTG TGA TGT CGA TGT CA G)/A CT1r (TTT GA G ATC CA C ATT TGT TGG AA); RNA as a template; and R O X r efer ence passiv e dy e follo wing the manufacturer's instructions as previously described (Dmytruk et al. 2020 ).

Biochemical analysis
The cell biomass of yeast was determined with a Helios Gamma spectrophotometer (OD, 590 nm; cuvette, 10 mm) turbidimetrically with gravimetric calibration.Flavin production was analyzed by measuring fluorescence (Turner Quantech FM 109510-33 fluor ometer, excitation maxim um at 440 nm, emission maxim um at 535 nm).
Concentrations of glucose and xylose in the medium broth wer e anal yzed by HPLC (PerkinElmer, Series 2000, USA) with an Aminex HPX-87H ion-exchange column (BioRad, Hercules, USA) and Refr activ e Index Detector (PerkinElmer, Series 200a, USA).A mobile phase of 4 mM H 2 SO 4 was used at a flow rate 0.6 mL min −1 and the column temper atur e was 30 • C.

Sta tistical anal ysis
All the experimental data shown in this manuscript were collected from at least three independent experiments to ensure reproducibility of the trends and relationships observed in the cultures.A statistical T-test was used.Eac h err or bar indicates the standard de viation (SD) fr om the mean obtained fr om the samples in biological triplicate .T he 5% significance le v el was used in the statistical analyses.

Riboflavin production by C. famata BRPI strain cultiv a ted in media with different carbon sources
A pr eliminary scr eening of carbon sources potentiall y pr esent in lignocellulosic hydr ol ysates supporting the growth and riboflavin production by C. famata was conducted.It was observed that BRPI accumulated biomass on hexoses (glucose , fructose , mannose, and galactose) in the range of 2.9 to 3.3 g L −1 , as well as on pentoses (xylose and L-arabinose) at 1.9 and 2.6 g L −1 , r espectiv el y (Table 1 ).Biomass accumulation on pentoses was a ppr oximatel y 25% lo w er compared to that on hexoses.All  tested carbon sources supported riboflavin production by BRPI.Riboflavin production and riboflavin production calculated per cell dry weight (CDW) on glucose or galactose were similar, reaching 812.5 or 757.5 mg L −1 and 248.5 or 265.8 mg g CDW −1 , respectiv el y (Table 1 ).Despite riboflavin production on xylose was only 337.5 mg L −1 the riboflavin production per CDW on this substrate was almost the same as on mannose reaching 170.4 mg g CDW −1 (Table 1 ).Although riboflavin production on xylose was only 337.5 mg L −1 , the riboflavin production per CDW on this substrate was nearly the same as that on mannose, reaching 170.4 mg g CDW −1 .Riboflavin production on L-arabinose was 1.46 times higher than that on xylose, r eac hing 492.5 mg L −1 (Table 1 ).
Since the main components of the lignocellulose hydr ol ysate are glucose and xylose (see "Material and methods"), we analyzed the riboflavin production of BRPI in a YNB medium contain-Table 3. Cell biomass , ribofla vin production, ribofla vin production per CDW and riboflavin yield of the recombinant strains BRPI/XYL1 and BRPI/XYL1/XYL2 of C. famata under conditions of high cell density cultivation in bioreactor in YNB medium containing five and three times diluted hydr ol ysate r epr esented at 120 h of cultiv ation.

Riboflavin production per CDW (mg g CDW
The strain studied achieved a biomass accumulation of up to 3.6 g L −1 in this medium.Riboflavin pr oduction r eac hed 772.5 g L −1 , with a riboflavin production per CDW of 234 mg g CDW −1 after 108 hours of cultivation.The yeast consumed glucose within 60 hours of cultivation and xylose within 108 hours.

Riboflavin production by strain BRPI C. famata , grown on media with lignocellulose hydrol ysa te
Biomass accumulation and riboflavin production by the C. famata str ain BRPI wer e assessed in media containing hydr ol ysate.BRPI was unable to grow in undiluted hydr ol ysate, likel y due to the presence of toxic concentrations of furfural, HMF and acetic acid.
Ther efor e, a modified medium containing diluted hydr ol ysate was emplo y ed.Synthetic YNB medium supplemented with 3 g L −1 ammonium sulfate, 2 g L −1 yeast extract, and four times diluted hydr ol ysate supported BRPI growth.
BRPI str ain ac hie v ed a biomass accumulation of up to 4.0 g L −1 in this medium (Fig. 3 ).Riboflavin production reached 432.5 g L −1 , with a riboflavin production per CDW of 109.4 mg g −1 of CDW after 108 hours of cultivation (Fig. 3 ).The yeast consumed completely glucose within 60 hours of cultivation and xylose in 108 hours of cultivation on diluted hydrolysate.It is important to note the simultaneous utilization of glucose and xylose from hydrolysate (Fig. 3 ).

Construction of recombinant strains of C. famata with expressed XYL1 and XYL1/XYL2 genes
As pr e viousl y observ ed (Table 1 ), the pr oduction of riboflavin in xylose was r elativ el y low compared to other sugars tested.To enhance xylose conversion to riboflavin, we opted to ov er expr ess the first genes of the xylose utilization pathway, XYL1 and XYL2 , which encode xylose reductase and xylitol dehydrogenase, respectively.The BRPI strain of C. famata was used as the parental one for overexpression of the XYL1 and XYL1/XYL2 genes.
The expression of the XYL1 gene was analyzed by qRT-PCR in BRPI/XYL1 strain on xylose and glucose as a control.It was found that the expression profiles of XYL1 on xylose was 3.8-fold increased as compared to that of the parental strain cultivated on xylose with no increase in glucose medium (Table 2 ).The expression of XYL1 and XYL2 in BRPI/XYL1/XYL2 strain on xylose increased by 15 and 15.6-fold, respectively, compared to that of BRPI.An increase in the expression of both these genes in glucose medium was visible, but to a lower extent than in xylose medium (Table 2 ).

Growth and riboflavin production by recombinant strains of C. famata with overexpressed XYL1 and XYL1/XYL2 genes
Characterization of riboflavin production in the recombinant strains of C. famata BRPI/XYL1 and BRPI/XYL1/XYL2 on the YNB medium with xylose was performed after 96 hours of cultivation.T he o v er expr ession of XYL1 and XYL1/XYL2 led to a slight increase in biomass accumulation, with the respective strains showing a 1.06-fold and 1.13-fold increase compared to BRPI (Fig. 4 , Table 2 ).The strain BRPI/XYL1 produced 450 mg L −1 of riboflavin, indicating a 1.36-fold increase of vitamin B2 production compared to that of BRPI (Fig. 4 , Table 2 ).T he ribofla vin production per CDW and riboflavin yield by BRPI/XYL1 were 211 mg g −1 of CDW and 23 mg g −1 of xylose, r espectiv el y.The str ain had a 1.28-fold increase in riboflavin production per g of CDW or riboflavin yield per g of consumed xylose compared to that of the par ental BRPI str ain (Table 2 ).Str ain BRPI/XYL1/XYL2 demonstrated 1.44-fold increase in riboflavin production that amounted to 474 mg L −1 (Fig. 4 , Table 2 ) when compared to the BRPI.T he ribofla vin production per CDW and ribofla vin yield of the BRPI/XYL1/XYL2 amounted to 210 mg g −1 of CDW and 24 mg g −1 of xylose .T he strain had a 1.28-fold or 1.33-fold increase in riboflavin production per g of CDW or riboflavin yield per g of consumed xylose when compared to the parental BRPI strain (Table 2 ).
The strain BRPI and BRPI/XYL1 were utilized for experiments in the bioreactor.Both were grown in YNB medium supplemented with five or three times diluted bagasse hydrolysate.Over the course of a 5-day bioreactor cultivation using a five times diluted hydr ol ysate, biomass accum ulation and riboflavin production by BRPI and BRPI/XYL1 strains reached 4.9 g L −1 and 5.2 g L −1 , and 780 mg L −1 and 900 mg L −1 , r espectiv el y (Fig. 5 A; Table 3 ).The biomass of yeast strains cultivated in a three times diluted hydr ol ysate was 1.6-fold higher compared to that in a five times diluted hydr ol ysate, r eac hing 8.2 g L −1 for both str ains (Table 3 ).Riboflavin production by BRPI and BRPI/XYL1 on this medium r eac hed 1356.2 mg L −1 and 1493.5 mg L −1 , r espectiv el y (Fig. 5 B; Table 3 ).Hence, the expression of XYL1 increased riboflavin production and riboflavin yield per consumed glucose and xylose by 15% and 10% in five and three times diluted hydrolysate, respectiv el y (Table 3 ).BRPI and BRPI/XYL1 completely consumed glucose within 48 hours of cultivation and xylose within 120 hours of cultivation, in a similar manner for both dilution rates.It is noteworthy that biomass accumulation and riboflavin production in three times diluted hydrolysate reached close to their peak values at 48 hours of culti vation, unlik e in five times diluted hydrolysate (Fig. 5 ).

Discussion
When considering the importance of c hemical feedstoc k, the pr oduction of high-value substances from renewable sources is becoming incr easingl y a ppealing.Lignocellulosic hydr ol ysates ar e attr activ e substr ates in micr obial fermentation pr ocesses for the pr oduction of commerciall y r ele v ant compounds (Ba ptista et al. 2021 ).Pr e vious r eports hav e demonstr ated riboflavin pr oduction from lignocellulosic hydrolysates by bacteria.Using corn cob hydr ol ysate as a carbon sour ce, gro wth and riboflavin biosynthesis w ere optimized b y the thermophilic strain Geobacillus thermoglucosidasius (Wang et al. 2022 ).The ov er expr ession of the mannose-6-phosphate isomerase gene manA , xylose isomerase gene xylA , and xylulokinase gene xylB to enhance mannose and xylose consumption in Corynebacterium glutamicum resulted in a 56% increase in riboflavin productivity from sugars derived from lignocellulose br eakdown (Pér ez-García et al. 2021 ).Howe v er, the riboflavin pr oduction by the mentioned micr oor ganisms r eac hed onl y 121 mg L −1 and 27 mg L −1 , which are too low for considering them as viable industrial producers of vitamin B2 at this stage.
So far, no micr oor ganism that could efficiently produce riboflavin in lignocellulosic hydr ol ysates has been reported.To address this gap, we tested the ability of the C. famata riboflavinov er pr oducing str ain BRPI, whic h was constructed in our pr e vious work (Dmytruk et al. 2020 ), to grow on the main sugars found in lignocellulosic hydr ol ysates and to pr oduce riboflavin.Both, hexoses (glucose , fructose , mannose , galactose) and pentoses (Larabinose, xylose) supported growth of BRPI (Table 1 ).Even more intriguing was the result that BRPI synthesized riboflavin on all tested carbon sources (Table 1 ).We have demonstrated, for the first time, that C. famata is capable of overproducing riboflavin on L-arabinose and xylose.It has been shown that L-arabinose is a good carbon source for vitamin B 2 production by recombinant Bacillus subtilis (Oraei et al. 2018 ).BRPI was also able to grow and produce riboflavin in synthetic medium with a glucose/xylose mixture and in medium with diluted sugarcane straw hydrolysate (Fig. 2 , 3 , 5 ).T he o v er expr ession of the XYL1 gene, encoding xylose r eductase, r esulted in a 10-15% increase in riboflavin production in medium with diluted hydr ol ysate.Ho w e v er, additional ov er expression of the XYL2 gene, which encodes xylitol dehydrogenase, did not lead to a further increase in riboflavin synthesis.The maximall y ac hie v ed riboflavin titer of ar ound 1.5 gr ams per liter show promise for future research and practical applications.
This work r epr esents the first comm unication, to the best of our knowledge, on the ov er pr oduction of riboflavin using lignocellulosic hydr ol ysate.Additional incr eases in riboflavin pr oduction could be attained through the two strategies involving strain construction: (i) enhancing riboflavin synthesis by activating pathways responsible for riboflavin precursors (GTP, ribulose-5-phosphate) synthesis, and (ii) further enhancing xylose utilization by ov er expr essing xylulokinase.It was observed that undiluted hydr ol ysate did not support the growth of C. f amata str ains, likely due to the inhibitory effects of furfural, HMF and acetic acid.This finding suggests the need for further work in selecting strains that are insensitive to these inhibitors.For instance, ada ptiv e labor atory e volution could be considered as a method to ac hie v e this goal (Ujor and Okonkwo 2022 ).Our results indicate that less diluted hydr ol ysate supports faster growth and higher riboflavin production (Fig. 5 ).Bearing this in mind, optimizing the fed-batch mode in bioreactors with hydrolysate as the carbon source could lead to increased riboflavin production.
Finally, it is worth emphasizing that C. famata is an ideal candidate for riboflavin production from lignocellulosic hydrolysates due to its robust growth and high riboflavin yield.Notably, it can efficiently utilize not only glucose and xylose but also other major sugars present in hydrolysates .T hese unique attributes , uncommon among nativ e micr oor ganism str ains, offer pr omising opportunities for the de v elopment of industrially viable riboflavin producers using lignocellulose, pectin, beet pulp, and other r ele v ant residues.

Conclusions
The engineer ed str ains of the yeast C. f amata ov er pr oduced riboflavin on lignocellulosic hydr ol ysate.Riboflavin accum ulation increased in the recombinant strains overexpressing the XYL1 and XYL2 genes, which code for xylose reductase and xylitol dehydrogenase, r espectiv el y.The highest riboflavin accumulation during bior eactor cultiv ation by BRPI/XYL1, using ba gasse hydr ol ysate as the carbon source, r eac hed 1.5 g L −1 .

Figure 2 .
Figure 2. Time profiles of growth (A), riboflavin production and riboflavin production per CDW (B) and sugars consumption (C) in flasks by strain BRPI C. famata on YNB supplemented with glucose and xylose in a ratio of 2.7 to 1 (16 g of glucose and 6 g of xylose).

Figure 3 .
Figure 3.Time profiles of growth (A), riboflavin production and riboflavin production per CDW (B) and sugars consumption (C) in flasks by strain BRPI C. famata using hydr ol ysate diluted 4 times with synthetic YNB medium supplemented with 2 g L-1 yeast extract.
using the primer pair Ko1258 (CGG GGT ACC AGT CTT A T A T A T A TC CGA ACT TGG)/Ko1259 (CCG GAA TTC TC A C AT AAC C A C AA G GTG CC).The KpnI and EcoRI restriction sites were incorporated into the Ko1258 and Ko1259 r espectiv el y.The SAT-1 was treated with KpnI and EcoRI, and dir ectl y cloned into the plasmid pUC57_prTEF1 Cf _trTEF1 Dh _Ble_Sa (Ruchala et al. 2022 ) digested with KpnI and EcoRI, instead of Ble_Sa.The resulting plasmid was designated as pT-SAT.
Ct values .T he error bars indicate standard de viations calculated fr om at least two independent experiments performed in triplicates .T he strains were grown in YNB medium supplemented with 20 g L −1

Figure 5 .
Figure 5. Production of riboflavin by C. famata yeast strains BRPI and BRPI/XYL1 in bioreactors on YNB medium with the addition of five times (A) or three times diluted hydrolysate (B).